Evaluation on Antioxidant Effect of Xanthohumol by Different Antioxidant Capacity Analytical Methods

Several assays have been frequently used to estimate antioxidant capacities including ABTS, DPPH, and FRAP assays. Xanthohumol (XN), the major prenylated flavonoid contained in beer, witnessed various reports on its antioxidant capacity. We systematically evaluated the antioxidant activity of XN using three systems, 2,2,-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) scavenging assays, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical assays, and ferric reducing antioxidant power (FRAP) assays.The results are expressed as Trolox equivalent antioxidant capacity (TEAC).The TEAC of XNwas 0.32±0.09 μmol⋅l by the ABTS assay and 0.27 ± 0.04 μmol⋅l by the FRAP. Meanwhile, the XN did not show obviously scavenging effect on DPPH radical reaction system. These results showed that different methods in the evaluation of compound antioxidant capicity, there may be a different conclusion.


Introduction
Antioxidant capacity analysis is an important indicator to evaluate the antioxidation of the component.Several assays have been frequently used to estimate antioxidant capacity in free radical biology including 2,2,-azino-bis-3ethylbenzthiazoline-6-sulphonic acid (ABTS •+ ) scavenging assays, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical assays, and ferric reducing antioxidant power (FRAP) assays.The difference of these methods is the use of different free radical.Xanthohumol (XN) is a natural prenylated chalcone derived from hops (Humulus lupulus L.).Over the past decade, anticancer, antimutagenic, and anti-inflammatory [1][2][3][4][5] properties of XN have been studied by many researchers, and all of these biological properties are based on its antioxidant effect.
ROS play a crucial role in the pathogenesis of several human diseases, such as cancer, rheumatoid (rheumatoid) arthritis, neurodegenerative diseases, and pulmonary diseases [6].It seems crucial to quench ROS as fast as possible before they attack biomolecules and cause harm.Antioxidants play an important role to eliminate ROS and other radicals; meanwhile, the antioxidant capacity is positively correlated with the ability of scavenging free radicals.There are many different kinds of evaluation reports for the antioxidant capacity of XN against ROS and other radicals.XN showed high antioxidant activity in inhibiting LDL oxidation [7], was able to scavenge reactive radicals including hydroxyl and peroxyl radicals, and inhibited superoxide anion and nitric oxide production [1].However, it has also been reported that XN to be prooxidant, was able to rapidly induces O 2 •− [8].In this paper, we compared with different methods for XN antioxidant capacity, to clarify whether methodology resulted in the conclusion differences.We have assessed the antioxidant capacity with attention to the following proposals: (1) Niki and Noguchi reported that there are two types of antioxidants that scavenge radicals quickly and quench many radicals, and they proposed assessing reactivity based on both reaction rate and stoichiometry [9]; (2) the activities of some antioxidants vary depending on the assay method, and thus use of multiple methods is recommended [9][10][11][12]; and (3) comparative studies using common antioxidants are essential to clarify the biological significance of the activities of sample.
Trolox equivalent antioxidant capacity (TEAC) assay has been widely accepted for assessing "antioxidant power" as its inexpensive, highly reproducible, straightforward, and speedy procedure [13][14][15][16].The TEAC method is based on the ability of antioxidant compounds to scavenge the longlived stable radical cation chromophore of 2,2-azinobis (3ethylbenzothiazoline 6-sulfonate; ABTS).Nowadays, TEAC value can be assigned to all compounds by comparing their scavenging capacity to that of Trolox, a water soluble vitamin E analogue [17], by several free radical reaction systems.
Xanthohumol was extracted from hop pellets (Humulus lupulus) in Yumen Tuopu Scientific and Technological Development Co.The briefly procedure as the following description.The purity was 98.64% by high performance liquid chromatography.

Measurement of XN Antioxidant
Capacity.Three types of radical scavenging capacity analytical methods were employed in the study as the following description.Each concentration of XN was run in triplicate, respectively.Mean and standard deviation ( = 3) were calculated.

𝐴𝐵𝑇𝑆 •+ Radical Cation Scavenging Activity of XN.
The assay is based on the inhibition of the absorbance of the radical cation of 2,2-azinobis (3-ethylbenzothiazoline 6-sulfonate; ABTS) which has characteristic long-wavelength absorption spectrum showing maximum at 734 nm [17,18] slightly modified by Erel [19].150 L of ABTS test reagent [17,18] and 50 L of sample were added to each well in a 96well microtiter plate.As unpaired electrons are sequestered by antioxidants in the sample, the ABTS test reagent turns colourless.The absorbance of reaction system at 734 nm with microplate reader is reduced.The percentage inhibition of ABTS •+ by the sample was calculated according to the following formula: 0 is the absorbance of the control, and   is the absorbance of the sample.The free radical scavenging capacity of the XN, calculated as percentage inhibition of ABTS •+ , was equated against a Trolox standard curve (10-60 mol⋅L −1 ).Results are expressed in TEAC (mol⋅L −1 ).Necessary dilution was done to ensure the ABTS value fell in the linear range of the standard curve.

𝐷𝑃𝑃𝐻
• Radical Scavenging Activity Assay of XN.The DPPH assay was performed according to the method developed by Brand-Williams et al. [20].The percentage inhibition of DPPH • by the sample was calculated according to the following formula: 0 is the absorbance of the control, and   is the absorbance of the sample.The free radical scavenging capacity of the XN, calculated as percentage inhibition of DPPH • , was equated against a Trolox standard curve (10-60 mol⋅L −1 ).Results are expressed in TEAC concentration (mol⋅L −1 ).Necessary dilution was done to ensure the DPPH value fell in the linear range of the standard curve.

Ferric-Ion Reducing Antioxidant Power.
A slightly modified FRAP method was used to test the total antioxidant capacity of XN [13].In Benzie and Strain's original FRAP protocol [13,21], 300 L of FRAP reagent and 40 L of sample were used in the reaction system.Half the amounts of FRAP reagent (150 L) and sample (20 𝜇L) were applied in this study to minimize the amount of sample needed to run the assay [22].Briefly, the FRAP reagent was produced by mixing 300 mmol⋅L −1 acetate buffer (pH 3.6), 10 mmol⋅L −1 TPTZ solution, and 20 mmol⋅L −1 FeCl 3 ⋅6H 2 O in a 10 : 1 : 1 ration and was prepared freshly at 37 ∘ C [21,23,24].A total of 150 L of working FRAP reagent and 20 L of sample were added into each well in a 96-well microtiter plate and incubated at room temperature for 30 min in dark [25].Reading of the colored product (ferrous tripyridyltriazine complex) was taken at 593 nm with microplate reader [22].The Trolox standard curve was linear between 10 and 60 mol⋅L −1 Trolox.The initial blank reading for each well with just FRAP reagent was then subtracted from the final reading of FRAP reagent with sample to determine the FRAP value for sample [25].Results are expressed in TEAC (mol⋅L −1 ).Necessary dilution was done to ensure the FRAP value fell in the linear range of the standard curve.Sample was measured in triplicate.Mean and standard deviation ( = 3) were calculated.

Statistical Analysis.
All data are presented as means (±SD) of at least three independent experiments, each experiment having a minimum of three replicates of sample.Student's paired -test, correlation analyses, and linear regression analyses were performed using SPSS (version 13).The level of statistical significance was set at  < 0.05 for two-side testing.

Results and Discussion
In the ABTS •+ radical cation scavenging assay system, the final concentrations of XN used in the analysis are 60, 50, 40, 30, 20, 10 mol⋅L −1 with 100% (v⋅v −1 ) ethanol, and there was significant linear correlation in the percentage inhibition of concentrations of the XN (15.1%-47%).The maximum inhibition was 47% at the concentration of XN being 60 mol⋅L −1 .The calibration curve (Figure 2) revealed a highly positive linear ( 2 = 0.999) correlation between the mean ABTS •+ inhibition percentage and Trolox concentration.This curve was therefore employed to reliably estimate antioxidant potential of the tested samples, and the curve revealed a highly positive linear ( 2 = 0.9821) correlation between mean ABTS •+ inhibition percentage and concentration of XN too (Figure 3).The TEAC value of XN was 0.32 ± 0.09 mol⋅L −1 by the ABTS assay (Table 1).DPPH • radical scavenging activity was quantified in terms of percentage inhibition of a preformed free radical by antioxidants in each sample.Trolox was used as standard antioxidant too.There was a highly positive linear Data are expressed as mean ± SD.Each assay was run three times ( = 3).( 2 = 0.9968) correlation between the mean inhibition value and the Trolox concentration (Figure 4).However, there was no statistic signification on the inhibition (10.7%-11.9%) of the DPPH • radical by the various concentrations of XN (250, 200, 150, 100, 50 mol⋅L −1 in 100% (v⋅v −1 ) ethanol) (Figure 5), with no clear linear correlation between the mean inhibition value and the XN concentration and no clear linear correlation between the mean inhibition value and the XN concentration ( 2 = 0.6668).
We evaluated the antioxidant capacity of XN by the TEAC assay through ABTS •+ , DPPH • , and FRAP system and found significant difference for reducing ability of tested sample.Results showed that XN has stronger antioxidant activity in the ABTS •+ free radical inhibition and FRAP assay system.In the ABTS •+ radical cation scavenging assay system, the 10 mol⋅L −1 of XN has 16.3% inhibition rate.The inhibition rate of XN reached 46.8% when its concentration was increased to 60 mol⋅L −1 .There was a highly positive linear ( 2 = 0.9839) correlation between mean inhibition value and concentrations of XN which showed that XN significantly   inhibited ABTS  [26,27].XN possesses OH phenolics in the B-ring.The Bors's found cannot be used to explain XN's weak DPPH free radical system scavenging effect therefore.
The farther studies are needed to clarify the relationship between the structure of XN and its free radical scavenging capacity in DPPH assay system.

Conclusion
The findings of the present study based on different analytical principals suggest that the ABTS, DPPH, and FRAP assays gave comparable results for the antioxidant activity measured of XN.XN is able to scavenge many types of ROS, has stronger free radical scavenging capacity in ABTS •+ than FRAP system, and has no or little scavenging effect on DPPH radical cation reaction.So, we need to pay attention to the choice of methods when evaluating the antioxidant capacity of XN.
•+ in a concentration-dependent manner.Since FRAP measures the reduction of FeIII-TPTZ to FeII-TPTZ, the reduction values were 49.2%, 42.3%, 37.2%, 31.5%, and 23.5% with final concentrations of XN (25, 50, 75, 100, and 125 mol⋅L −1 ).Comparing ABTS •+ with FRAP radical assay system, XN has stronger scavenging capacity in ABTS •+ than FRAP system.In the DPPH assay system, XN showed that the maximum inhibitory is 11.9% with any concentration of XN.Therefore, XN has no or little scavenging effect on DPPH radical cation.Danila et al. have reported that a DPPH does not react with flavonoids without substituted OH in the B-ring or with aromatic acids with a single OH group; consequently, it could be said that DPPH scavenging highly depends on the degree of electron delocalization in a structure-activity study